This proposal is focused on vinculin, a cytoskeletal protein that is a prominent component of focal adhesions and adherens junctions. Vinculin exists in an autoinhibited conformation and upon activation, functions as a scaffold to regulate cellular events resulting in cell migration, cell survival and embryogenesis. Vinculin null cells display tumorigenic properties and mutation or loss of vinculin is associated with cardiac disease. Although vinculin binds actin and phosphatidylinositol 4,5- bisphosphate (PIP2), we do not understand the nature of these interactions or their precise role in regulating vinculin function. In particular, the interaction between vinculin and actin plays a pivotal role in linking transmembrane receptors to the cytoskeleton, which, in turn, is important for controlling cellular cell morphology, force transmission and motility. Vinculin binds to F-actin and undergoes a conformational change that induces formation of a cryptic dimer necessary for actin filament bundling, but the conformation change that occurs and dimer that is formed is unknown. It is also unclear how vinculin recognizes PIP2, inserts into membranes and is regulated by this interaction. We propose highly integrated computational and experimental approaches to generate and test models for these important interactions and assess their significance in vinculin function both in vitro and in cells. This will be accomplished by generating and characterizing vinculin variants with specific defects in actin binding, actin-induced vinculin dimer formation and PIP2 association in vitro, and then expressing the full length wild type protein and mutants in vinculin null cells. The role of these interactions in regulating the activation state of vinculin as well as vinculin's force response and transmission properties will be probed at both the sub-cellular and whole cell level.